Detection of oxidative damage by 32P-postlabelling: 8-hydroxydeoxyguanosine as a marker of exposure.
AffiliationCancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
MetadataShow full item record
AbstractHuman exposure to reactive oxygen species is unavoidable and has been implicated in the etiology of a number of human diseases. This exposure results in the formation of various modified DNA bases: the promutagenic lesion 8-hydroxydeoxyguanosine (8OHdG), in particular, is a major product. We have developed an assay using ion-pair HPLC and 32P-postlabelling to quantify 8OHdG in human DNA with high specificity and sensitivity. An internal standard is used to account for variations in labelling efficiency. Chemically synthesized 8OHdG 3'-monophosphate and 5'-monophosphate standards were used to optimize the HPLC-32P-postlabelling and TLC separative steps, respectively. The assay was validated using known ratios of 8OHdG to normal nucleotides. The limit of detection is in the range of one 8OHdG residue per 10(6)-10(7) dG residues. Using this procedure, 8OHdG levels of 16-35 8OHdG adducts per 10(5) dG residues have been found in leukocytes isolated from patients who received 180-200 cGy of ionizing radiation. These levels were 2-4-fold greater than those found in an unexposed individual. Since 8OHdG may be formed during DNA extraction and digestion, current procedures for measuring background levels are discussed.
CitationDetection of oxidative damage by 32P-postlabelling: 8-hydroxydeoxyguanosine as a marker of exposure. 1993 (124):105-14 IARC Sci. Publ.
JournalIARC Scientific Publications
- Combined micropreparative techniques with synchronous fluorescence spectroscopy or 32P-postlabelling assay for carcinogen-DNA adduct determination.
- Authors: Shields PG, Kato S, Bowman ED, Petruzzelli S, Cooper DP, Povey AC, Weston A
- Issue date: 1993
- 8-Hydroxydeoxyguanosine as a urinary biomarker of oxidative DNA damage.
- Authors: Loft S, Fischer-Nielsen A, Jeding IB, Vistisen K, Poulsen HE
- Issue date: 1993 Oct-Nov
- 32P-postlabelling methods for cyclic DNA adducts.
- Authors: Watson WP, Crane AE, Steiner S
- Issue date: 1993
- Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index.
- Authors: Loft S, Vistisen K, Ewertz M, Tjønneland A, Overvad K, Poulsen HE
- Issue date: 1992 Dec
- 32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2'-deoxyguanosine.
- Authors: Zeisig M, Hofer T, Cadet J, Möller L
- Issue date: 1999 Jul